1. Field of the Invention
The present invention relates to methods and materials in the field of molecular biology and the regulation of protein synthesis through plant genetic engineering. More particularly, the invention relates to the introduction of a foreign nucleotide sequence into a plant genome, wherein the introduction of the nucleotide sequence effects an increase in the syringyl content of lignin synthesized by the plant. Specifically, the invention relates in one aspect to methods for modifying the lignin composition in a plant cell by the introduction thereinto of a foreign nucleotide sequence comprising a tissue-specific plant promoter sequence and a coding sequence encoding an active ferulate-5-hydroxylase (F5H) enzyme. Plant transformants harboring an inventive promoter-F5H construct demonstrate increased levels of syringyl monomer residues in lignin synthesized thereby, rendering the polymer more readily delignified and, thereby, rendering the plant more readily pulped or digested.
2. Discussion of Related Art
Lignin is one of the major products of the general phenylpropanoid pathway, set forth in FIG. 1, and is one of the most abundant organic molecules in the biosphere (Crawford, (1981) Lignin Biodegradation and Transformation, New York: John Wiley and Sons). Referring to FIG. 1, lignin biosynthesis via the phenylpropanoid biosynthetic pathway is initiated by the conversion of phenylalanine into cinnamate through the action of phenylalanine ammonia lyase (PAL). The second enzyme of the pathway is cinnamate-4-hydroxylase (C4H), a cytochrome P450-dependent monooxygenase (P450) which is responsible for the conversion of cinnamate to p-coumarate. The second hydroxylation of the pathway is catalyzed by a relatively ill-characterized enzyme, p-coumarate-3-hydroxylase (C3H), whose product is caffeic acid. Caffeic acid is subsequently O-methylated by caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT) to form ferulic acid, a direct precursor of lignin. The last hydroxylation reaction of the general phenylpropanoid pathway is catalyzed by F5H. The 5-hydroxyferulate produced by F5H is then O-methylated by OMT, the same enzyme that carries out the O-methylation of caffeic acid. This dual specificity of OMT has been confirmed by the cloning of the OMT gene, and expression of the protein in E. coli (Bugos et al., Plant Mol. Biol. 17, 1203, (1991); Gowri et al., (1991) Plant Physiol., 97, 7, (1991)).
Recently, a different route for the biosynthesis of lignin monomers has received attention (Kneusel et al., Arch. Biochem. Biophys. 269, 455, (1989); Kxc3xchnl et al., Plant Science 60, 21, (1989); Pakusch et al., Arch. Biochem. Biophys. 271, 488, (1989); Pakusch et al., Plant Physiol. 95, 137, (1991); Schmitt et al., Jour. Biol. Chem. 266, 17416, (1991); Ye et al., Plant Cell 6, 1427, (1994); Ye and Varner, Plant Physiol. 108, 459, (1995)). This so-called xe2x80x9calternativexe2x80x9d pathway involves the activation of p-coumaric acid to its coenzyme A thioester, followed by hydroxylation and methylation reactions that generate feruloyl-CoA as the product of the pathway. Considering that ferulic acid can also be synthesized by the free acid pathway and can be activated to its CoA thioester by (hydroxy)cinnamoyl CoA ligate (4CL), lignin monomer biosynthesis probably occurs via a cross-linked network of pathways. Indeed, the continued accumulation of guaiacyl lignin in OMT suppressed plants (Atanassova et al., Plant J. 8, 465, (1995) 1995; Van Doorsselaere et al., Plant J. 8, 855, (1995)) indicates that the alternative pathway may be a major contributor to lignin biosynthesis in woody plants. Both the conventional xe2x80x9cfree acidxe2x80x9d pathway and the xe2x80x9calternativexe2x80x9d pathway have been reported to be developmentally regulated, providing different routes for the synthesis of lignin monomers in different cell types (Ye and Varner, supra). This differential gene regulation may be one of the mechanisms by which lignin monomer composition is controlled.
The committed steps of lignin biosynthesis are catalyzed by (hydroxy)cinnamoyl CoA reductase (CCR) and (hydroxy)cinnamoyl alcohol dehydrogenase (CAD), which ultimately generate coniferyl alcohol from ferulic acid and sinapoyl alcohol from sinapic acid. Coniferyl alcohol and sinapoyl alcohol are polymerized by extracellular oxidases to yield guaiacyl lignin and syringyl lignin respectively, although syringyl lignin is more accurately described as a co-polymer of both monomers.
Although ferulic acid, sinapic acid, and in some cases p-coumaric acid are channeled into lignin biosynthesis, in some plants these compounds are precursors for soluble secondary metabolites. For example, in Arabidopsis, sinapic acid serves as a precursor for lignin biosynthesis but it also channeled into the synthesis of soluble sinapic acid esters. In this pathway, sinapic acid is converted to sinapoylglucose which serves as an intermediate in the biosynthesis of sinapoylmalate (FIG. 1). Sinapic acid and its esters are fluorescent and may be used as a marker of plants deficient in those enzymes needed to produce sinapic acid (Chapple et al., Plant Cell 4, 1413, (1992)).
In nature, lignification, or integration of lignin into the plant secondary cell wall, provides rigidity and structural integrity to wood and is in large part responsible for the structural integrity of tracheary elements in a wide variety of plants, giving them the ability to withstand tension generated during transpiration. Lignin also imparts decay resistance to the plant secondary cell wall and is thought to have been essential to the evolution of terrestrial plants. Lignin is well suited to these capacities because of its physical characteristics and its resistance to biochemical degradation. Unfortunately, this same resistance to degradation has a significant impact on the utilization of lignocellulosic plant material (Whetten et al, Forest Ecol. Management 43, 301, (1991)).
In angiosperms, lignin is composed mainly of two aromatic monomers which differ in their methoxyl substitution pattern. As described above, precursors for lignin biosynthesis are synthesized from L-phenylalanine via the phenylpropanoid pathway which provides ferulic acid (4-hydroxy-3-methoxycinnamic acid) and sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) for the synthesis of guaiacyl- and syringyl-substituted lignin monomers, respectively. Two cytochrome P450-dependent monoxygenases (450s) are required for the synthesis of lignin monomers. C4H catalyzes the second step of the phenylpropanoid pathway, the hydroxylation of the aromatic ring of cinnamic acid at the para position, and its activity is required for the biosynthesis of all lignin precursors. Ferulate-5-hydroxylase (F5H) catalyzes the meta-hydroxylation of ferulic acid in the monomer-specific pathway branch required for sinapic acid an syringyl lignin biosynthesis.
The balance between guaiacyl and syringyl units in lignin varies between plant species, within a given plant, and even within the wall of a single plant cell. For example, the lignin of the mature Arabidopsis rachis (flowering stem) contains guaiacyl and syringyl residues in an overall ratio of approximately 4:1; however, this ratio is not constant throughout plant development. The syringyl content of the rachis increases from less than 6 mol % within the apical 4 cm of the bolt to over 26 mol % near the base of the inflorescence. Histochemical staining of Arabidopsis rachis cross-sections indicates that syringyl lignin biosynthesis is also developmentally regulated in a tissue-specific manner. Accumulation of syringyl lignin (i.e., lignin synthesized from syringyl and guaiacyl monomers) is restricted to the cells of the sclerified parenchyma that flank the vascular bundles while guaiacyl lignin (i.e. lignin synthesized from guaiacyl monomers only) is deposited only in the cells of the vascular bundle. The increase in syringyl lignin content during rachis development is a consequence of sclerified parenchyma maturation as these cells undergo secondary thickening after the vascular bundle has been formed from the cells of the procambium.
The monomeric composition of lignin has significant effects on its chemical degradation during industrial pulping (Chiang et al., Tappi, 71, 173, (1988). The guaiacyl lignins (derived from ferulic acid) characteristic of softwoods such as pine, require substantially more alkali and longer incubations during pulping in comparison to the guaiacyl-syringyl lignins (derived from ferulic acid and sinapic acid) found in hardwoods such as oak. The reasons for the differences between these two lignin types has been explored by measuring the degradation of model compounds such as guaiacylglycerol-guaiacyl ether, syringylglycerol-guaiacyl ether, and syringylglycerol-(4-methylsyringyl) ether (Kondo et al., Holzforschung, 41, 83, (1987)) under conditions that mimic those used in the pulping process. In these experiments, the mono- and especially di-syringyl compounds were cleaved three to fifteen times faster than their corresponding diguaiacyl homologues. These model studies are in agreement with studies comparing the pulping of Douglas fir and sweetgum wood where the major differences in the rate of pulping occurred above 150 C. where arylglycerol-aryl ether linkages were cleaved (Chiang and Funaoka, Holzforschung, 44, 309, (1990)).
Another factor affecting chemical degradation of the two lignin forms may be the condensation of lignin-derived guaiacyl and syringyl residues to form diphenylmethane units. The presence of syringyl residues in hardwood lignins leads to the formation of syringyl-containing diphenylmethane derivatives that remain soluble during pulping, while the diphenylmethane units produced during softwood pulping are alkali-insoluble and thus remain associated with the cellulosic products (Chiang et al., Holzforschung, 44, 147, (1990); Chiang and Funaoka, supra). Further, it is thought that the abundance of 5-5xe2x80x2-diaryl crosslinks that can occur between guaiacyl residues contributes to resistance to chemical degradation. This linkage is resistant to alkali cleavage and is much less common in lignin that is rich in syringyl residues because of the presence of the 5-O-methyl group in syringyl residues. Thus, the incorporation of syringyl residues results in what is known as xe2x80x9cnon-condensed ligninxe2x80x9d, a polymer that is significantly easier to pulp than condensed lignin.
Similarly, lignin composition and content in grasses is a major factor in determining the digestibility of lignocellulosic materials that are fed to livestock (Jung, H. G. and Deetz, D. A. (1993) Cell wall lignification and degradability in Forage Cell Wall Structure and Digestibility (H. G. Jung, D. R. Buxton, R. D. Hatfield, and J. Ralph eds), ASA/CSSA/SSSA Press, Madison, Wis.) The incorporation of the lignin polymer into the plant cell wall prevents microbial enzymes from having access to the cell wall polysaccharides that make up the plant cell wall. As a result, these polysaccharides are substantially unavailable for digestion by livestock, and much of the valuable carbohydrates contained within animal feedstock passes through the animals undigested. Thus, an increase in the dry matter of grasses over the growing season is counteracted by a decrease in digestibility causes principally by increased cell wall lignification. In light of the above background, it is clear that biotechnological modification or manipulation of lignin monomer composition is economically desirable, as it provides the ability to significantly decrease the cost of pulp production and to increase the nutritional value of animal feed stocks thereby also enhancing their economic value.
The mechanism(s) by which plants control lignin monomer composition has been the subject of much speculation. As mentioned above, gymnosperms do not synthesize appreciable amounts of syringyl lignin. In angiosperms, syringyl lignin deposition is developmentally regulated: primary xylem contains guaiacyl lignin, while the lignin of secondary xylem and sclerenchyma is guaiacyl-syringyl lignin (Venverloo, Holzforschung 25, 18 (1971); Chapple et al., supra). No plants have been found to contain purely syringyl lignin. It is still not clear how this specificity is controlled; however, a number of enzymatic steps have previously been proposed as sites for the control of lignin monomer compositions and at least five possible enzymatic control sites exist, namely OMT, F5H, 4CL, CCR, and CAD. For example, the substrate specificities of OMT (Shimada et al., Phytochemistry, 22, 2657, (1972); Shimada et al., Phytochemistry, 12, 2873, (1973); Gowri et al., supra; Bugos et al., supra) and CAD (Sarni et al., Eur. J. Biochem., 139, 259, (1984); Goffner et al., Planta., 188, 48, (1992); O""Malley et al., Plant Physiol., 98, 1364, (1992)) are correlated with the differences in lignin monomer composition seen in gymnosperms and angiosperms, and the expression of 3CL isozymes (Grand et al., Physiol. Veg. 17, 433, (1979); Grand et al., Planta., 158, 255, (1983)) has been suggests to be related to the tissue specificity of lignin monomer composition seen in angiosperms.
Although there are at least five possible enzyme targets, much attention has been directed recently to investigating the use of OMT and CAD to manipulate the lignin monomer composition in transgenic plants (Dwivedi et al., Plant Mol. Biol. 26, 61, (1994); Halpin et al., Plant J. 6, 339, (1994); Ni et al., Transgen, Res. 3, 120 (1994). Atanassova et al., supra: Van Doorsselaere et al., supra). Most of these studies have focused on sense and antisense suppression of OMT expression. This approach has met with variable results, probably owing to the degree of OMT suppression achieved in the various studies. The most dramatic effects were seen by using homologous OMT constructs to suppress OMT expression in tobacco (Atanassova et al., supra) and poplar (Van Doorsselaere et al., supra). Both of these studies found that as a result of transgene expression, there was a decrease in the content of syringyl lignin and a concomitant appearance of 5-hydroxyguaiacyl residues. As a result of these studies, Van Doorsselaere et al., (WO 9305160) disclose a method for the regulation of lignin biosynthesis through the genomic incorporation of an OMT gene in either the sense of anti-sense orientation. In contrast, Dixon et al. (WO 9423044) demonstrate the reduction of lignin content in plants transformed with an OMT gene, rather than a change in lignin monomer composition.
Similar research has focused on the suppression of CAD expression. The conversion of coniferaldehyde and sinapaldehyde to their corresponding alcohols in transgenic tobacco plants has been modified with the incorporation of an A. cordata CAD gene in anti-sense orientation (Hibino et al., Biosci. Biotechnol. Biochem. 59, 929, (1995)). A similar effort aimed at antisense inhibition of CAD expression generated a lignin with increased aldehyde content, but only a modest change in lignin monomer composition (Halpin et al, supra). This research has resulted in the disclosure of methods for the reduction of CAD activity using sense and anti-sense expression of a cloned CAD gene to effect inhibition of endogenous CAD expression in tobacco [Boudet et al., (U.S. Pat. No. 5,451,514) and Walter et al., (WO 9324638); Bridges et al., (CA 2005597)]. None of these strategies, however, increased the syringyl content of lignin, a trait that is correlated with improved digestibility and chemical degradability of lignocellulosic material (Chiang et al., supra. Chiang and Funaoka, supra; Jung et al., supra).
In view of this background, the present invention involves producing transformed plants having increased levels of syringyl residues in their lignins to facilitate chemical degradation of the lignin. Increased syringyl content in lignin produced by a plant transformed in accordance with the invention is achieved by modifying the enzyme pathway responsible for the production of lignin monomers in a manner distinct from those attempted previously. Specifically, this result is achieved in one preferred aspect of the invention by eliciting over-expression of the enzyme F5H in plant cells undergoing lignin synthesis. The term xe2x80x9cexpressionxe2x80x9d, as used herein, refers to the production of the protein product encoded by a nucleotide coding sequence. xe2x80x9cOver-expressionxe2x80x9d refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
Although F5H is a key enzyme in the biosynthesis of syringyl lignin monomers it has not been exploited to date in efforts to engineer lignin quality. In fact, since the time of its discovery over 30 years ago (Higuchi et al., Can. J. Biochem. Physiol., 41, 613, (1963)) there has been only one demonstration of the activity of F5H published (Grand, C., FEBS Lett. 169, 7, (1984)). Grand demonstrated that F5H from poplar was a cytochrome P450-dependent monooxygenase (P450) as analyzed by the classical criteria of dependence of NADPH and light-reversible inhibition by carbon monoxide. Grand further demonstrated that F5H is associated with the endoplasmic reticulum of the cell. The lack of attention given to F5H in recent years may be attributed in general to the difficulties associated with dealing with membrane-bound enzymes, and specifically to the liability of F5H when treated with the detergents necessary for solubilization (Grand, supra). The most recent discovery surrounding F5H has been made by Chapple et al., (supra) who reported a mutant of Arabidopsis thaliana L. Heynh named fah1 that is deficient in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. This locus, termed FAH1, encodes F5H.
In spite of sparse information about F5H in the published literature, the present inventor has been successful in the isolation, cloning, and sequencing of the F5H gene (Meyer et al., Proc. Natl. Acad. Sci. USA 93, 6869(1996)). The present inventor has also demonstrated that the stable integration of the F5H gene into the plant genome, where the expression of the F5H gene is under the control of a promoter other than the gene""s endogenous promoter (such as, for example, the 35S promoter), leads to an altered regulation of lignin biosynthesis. It has been determined that causing over-expression of the enzyme F5H in Arabidopsis using the 35S promoter allows the plant to produce lignin containing up to 30% of the syringyl monomer. This over-expression may be accomplished by constructing a 35S promoter/F5H construct and transforming a plant host with the construct. Similarly, over-expression of the enzyme F5H in tobacco using the 35S promoter allows the plant to produce lignin in its petioles (leaf stems) containing up to 40% of the syringyl monomer. One problem with this system, however, is that Arabidopsis plants transformed with the construct are unable to produce lignin having syringyl content greater than about 30mol %. Similarly, in tobacco plants transformed with the 35S promoter/F5H construct, no change was observed in the syringyl monomer content of stem lignin which is naturally approximately 50%.
These limitations are overcome by the present invention, which provides in one preferred aspect a genetic construct assembled from a tissue-specific promoter sequence endogenous to plant cells and a nucleotide sequence which encodes the enzyme F5H. The construct may be used to transform plants, thereby providing transformed plants capable of producing lignin having a syringyl content greater than a native plant. For example, an Arabidopsis plant may be transformed in accordance with the invention such that the transformed Arabidopsis plant is capable of producing lignin having syringyl content of greater than about 30 mol %. Furthermore, inventive constructs may be used to transform a tobacco plant such that the transformed tobacco plant is capable of producing lignin in its petioles having a syringyl content of greater than about 40 mol % and such that the transformed tobacco plant is capable of producing stem lignin having a syringyl content of greater than about 50 mol %.
The present invention relates to the isolation, purification and use of DNA constructs comprising a tissue-specific plant promoter, for example, a C4H promoter, and a nucleotide sequence useful for the modification of lignin biosynthesis such as, for example, an F5H coding sequence. Inventive DNA constructs employing lignification-specific promoters such as the C4H promoter are useful for modifying the quality or quantity of a plants lignin, and specific examples of constructs are provided herein for increasing the syringyl content of a plant""s lignin by targeting over-expression of the F5H enzyme to plant cells producing lignin or providing the precursors for lignin biosynthesis. Lignification-specific promoters set for in FIG. 1, such as the C4H promoter are effective in directing gene expression to lignifying cells, and are thus useful promoters for modifying gene expression in these cells via antisense or co-suppression technologies. As discussed in the Background above and set forth in FIG. 1, the F5H enzyme catalyzes an irreversible hydroxylation step that diverts ferulic acid away from the guaiacyl lignin biosynthesis and toward sinapic acid and syringyl lignin biosynthesis. Specifically, F5H catalyzes the reaction of ferulate to 5-hydroxyferulate and over-expression thereof in the proper plant tissues under the control of lignification-specific promoters such as the C4H promoter results in synthesis of lignin having a high syringyl content, i.e., greater than that achieved in prior art plants of the same species.
High syringyl lignins are more readily degraded during the pulping process and during ruminant digestion of lignocellulosic feedstocks. The unaltered morphology of tracheary elements and sclerified parenchyma in transgenic plants depositing lignin highly enriched in syringyl units suggests that this lignin still provides lignified cells with sufficient rigidity to function normally in water conduction and mechanical support. Thus, a surprisingly advantageous result is achieved in accordance with the invention upon increasing the syringyl content of crop species and trees, thereby generating lignins that are easier to digest or extract without detrimental consequences on agricultural performance.
It is presently shown that inventive DNA constructs may advantageously be used according to the invention to transform a plant, thereby providing an inventive transformed plant which produces lignin having a syringyl:guaiacyl ratio that is greater than that of a non-transformed plant of the same species or a plant of the same species transformed using constructs known in the prior art. The present invention thus provides methods for genetically engineering plants to provide inventive transformed plants which may be readily delignified. The invention features DNA constructs comprising a tissue-specific plant promoter sequence and a coding sequence as set forth herein, as well as DNA constructs comprising nucleotide sequences having substantial identity thereto and having similar levels of functionality. Inventive constructs may be inserted into an expression vector to produce a recombinant DNA expression system which is also an aspect of the invention.
In a preferred aspect of the invention, there is provided an isolated nucleic-acid construct comprising a nucleotide sequences which correspond to a regulatory sequence of the C4H genomic sequence set forth in SEQ ID NO:1 and a nucleotide sequence having substantial similarity to the sequence set forth in either SEQ ID NO:2 (F5H genomic nucleotide sequence) or SEQ ID NO:3 (F5H cDNA). In a preferred aspect of the invention, the enzyme encoded thereby preferably has an amino acid sequence having substantial identity to the F5H enzyme set forth in SEQ ID NO:4, wherein the amino acid sequence may include amino acid substitutions, additions and deletions that do not alter the function of the F5H enzyme.
It is an object of the present invention to provide an isolated DNA construct which comprises a tissue-specific promoter and a nucleotide sequence encoding an F5H enzyme, the construct finding advantageous use when incorporated into a vector or plasmid as a transformant for a plant.
Additionally, it is an object of the invention to provide transformed plants which produce lignin having a syringyl content grater than a native plant of the same species, thereby providing resources for the pulping industry which are much more readily and economically delignified, and providing agricultural feedstocks which are much more readily and efficiently digested by livestock.
Further objects, advantages and features of the present invention will be apparent from the detailed description herein.